Isolated cocoon silk protein from Simulium vittatum and nucleic acids encoding such protein

ABSTRACT

An isolated nucleic acid molecule encoding the cocoon silk protein from the black fly, Simulium vittatum. Also provided are the amino acid sequence derived from the cocoon silk, primers used to screen cDNA libraries to promote the building of a complimentary strand of DNA encoding the cocoon silk protein, a transformed microorganism containing cDNA which codes for cocoon silk protein, the amino acid sequence translated from the isolated gene of the cocoon silk (deduced from nucleotide sequence), primers for constructing a segment of recombinant DNA.

This application claims priority from U.S. Provisional Application No.60/214,992, filed Jun. 29, 2000 which is herein incorporated byreference in its entirety.

The sequence listing information submitted on computer readable form isidentical to the written sequence listing contemporaneously submitted onpaper, and includes no new matter.

FIELD OF THE INVENTION

The present invention relates to the cocoon silk protein isolated fromthe black fly, Simulium vittatum.

BACKGROUND OF THE INVENTION

Silk is a natural, protein filament fiber. Several types of natural silkthat are known to date are excreted by invertebrates such as those thatbelong to two classes of the phylum Arthropoda: Insecta and Arachnida.Silk producing insects include silk worms, black flies, wasps, andlacewing flies.

Some arthropods' silk have now been cloned. For example, Lewis, R. V. etal. (U.S. Pat. No. 5,728,810) teach the preparation of spider silkprotein by recombinant DNA techniques. Lewis, R. V., et al. (U.S. Pat.No. 5,733,771) teach a cDNA encoding minor ampulate silk proteins.Lombardi, S. J. et al. (U.S. Pat. No. 5,245,012) teach a recombinantspider silk protein which can be obtained in a commercially useful formby the cloning of host cells encoding such protein.

Another silk producing arthropod, the black fly, evolved to produce avery durable silk filament. Silk is produced by the black fly “larva”which forms a cocoon. The larva and pupae are aquatic but are confinedto running waters where they attach themselves to firm substrates. Theblack fly's silk filament is able to withstand the exposure to waterflow in order to keep the pupa inside the cocoon intact. Anotherremarkable property of the Simuliidae silk is its ability to maintainits adhesive characteristic while submerged in water. These propertiesare very attractive in terms of possible application of the black flysilk as a biomaterial.

The above prior art references are incorporated herein by reference.

The prior art does not teach the isolation of a nucleic acid moleculecoding for the silk protein from black flies. Further, the prior artdoes not teach the expression of such silk protein using recombinant DNAtechniques.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides an isolatedpolypeptide molecule having an amino acid sequence of SEQ ID NO: 1. In afurther embodiment, the present invention provides an isolated nucleicacid molecule coding for such polypeptide. In another embodiment, thepresent invention provides the nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO: 6. In a further embodiment, thepresent invention provides a polypeptide molecule having an amino acidsequence of SEQ ID NO: 7 and expressed by such nucleic acid molecule. Inyet another embodiment, the present invention provides an isolatednucleic acid molecule coding for such polypeptide. In addition, thepresent invention provides a cloning vector comprising the nucleotidesequence of SEQ ID NO: 6 and a host cell transformed with such vector.In a further embodiment, the present invention provides a fiber formedfrom polypeptide of SEQ ID NO: 7. In yet another embodiment, the presentinvention provides a method of isolating cocoon silk protein comprisingthe steps of: a) boiling a cocoon in a sample reducing buffer to removeSDS (sodium dodecyl sulfate)-soluble proteins, b) centrifuging thesample, c) withdrawing supernatant, adding formic acid to the pellet andincubating the sample in order to solubilize SDS-insoluble proteins, d)freezing and lyophilizing the sample in order to freeze-dry the sample,e) re-suspending the dried sample in a buffer to protect proteinsagainst potential residual proteolytic activity for subsequent analysisusing SDS-PAGE (polyacrylamide gel electrophoresis).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In a preferred embodiment, the present invention provides an isolatednucleic acid molecule encoding the silk protein of black fly cocoons.The invention also provides amino acid molecules expressed by suchnucleic acid as well as cells transformed with such nucleic acidmolecule. Also provided are primers for screening DNA libraries for theDNA encoding the subject silk protein.

Methods and Results of Research

1. Method of Isolating and Purifying Cocoon Silk Protein from Simuliumvittatum

The following method was developed for isolating and purifying cocoonsilk protein from the black fly, Simulium vittatum. In the preferredembodiment, the method comprises the following procedure. A singlecocoon from S. vittatum was boiled in 500 μl sample reducing buffer forone minute and then centrifuged for one minute at 13,000 rpm. Boiling insample reducing buffer removes any SDS-soluble proteins. The compositionof sample reducing buffer is as follows:

1 mL 0.5M Tris-HCl, pH 6.8

0.8 mL glycerol

1.6 mL

10% SDS (sodium dodecyl sulfate)

0.4 mL 2β mercaptoethanol

0.2 mL 0.5% bromophenol blue

4 mL H₂O

The supernatant was then removed and the sample was washed 4 times withdH₂O by spinning the sample down and pouring off water between washes.Then the pellet was re-suspended in 500 μl of 90% formic acid and thesample was incubated in a shaker at 22° C. for 1 hour in order tosolubilize the SDS-insoluble proteins. The sample was then frozen andlyophilized in order to be freeze-dried. Then the sample (formic acidand cocoon) was transferred to a 50 mL centrifuge tube and the volumewas increased to 50 mL by the addition of dH₂O. This centrifuge tube wasthen frozen in a −70° C. freezer and the sample was lyophilized. Afterthat, the sample was re-suspended in 400 μl of TEPI. TEPI buffercontains:

10 mM Tris-HCl, pH 8.0

1 mM EDTA (ethylenediaminetetraacidic acid)

1 μM phenylmethylsulfonylfluoride (PMSF)

100 μM iodoacetamide

Re-suspension in TEPI protected proteins against potential residualproteolytic activity for subsequent analysis using SDS polyacrylamidegel electrophoresis (SDS-PAGE). The sample was then run on aSDS-polyacrylamide gel in duplicate using standard procedures outlinedin Laemmli (Cleavage of Structural Proteins During the Assembly of theHead of Bacteriophage T4, Nature, 1970, 227:680-685, the content ofwhich we incorporate herein by reference). One gel was silver stainedand the other was transferred to a poly-vinylidene-difluoride (PVDF)membrane which was stained with Ponceau stain. The band on the gel thatcorresponded to the cocoon silk protein of S. vittatum was excised usinga razor blade and sent to the Centre de Recherche du CHUL (Quebec,Canada) for N-terminal amino acid sequencing.

2. N-terminal Amino Acid Sequence for Black Fly Cocoon Silk

The N-terminal amino acid sequencing of the silk protein isolated aboverevealed the following sequence:

GVAPKKYRKGHYVGGYGKKY SEQ ID NO: 1

3. cDNA Construction

In the preferred embodiment, cDNA was constructed as follows. Salivaryglands were dissected from 10 S. vittatum larvae and placed into anRNAse free Eppendorf tubule, on ice. After that, 1 mL of TRIZOL™ reagent(Life Technologies Inc.) was added. Total RNA was recovered usingmanufacturer's instructions.

Poly A⁺ mRNA was then isolated from the total RNA using Qiagen'sOligotex™ mRNA Kit. Oligotex provides a hybridization carrier on whichnucleic acids containing polyadenylic acid sequences can be simply andefficiently immobilized and easily recovered. Briefly, the Oligotexprocedure for isolation and purification of poly A⁺ mRNA takes advantageof the fact that most eukaryotic mRNAs end in a homopolymer of 20-250adenosine nucleotides, known as the poly A tail. The poly A tail isadded to the RNA transcript in the nucleus following transcription. Incontrast, structural RNAs are not polyadenylated. Nuclearpolyadenylation of mRNAs performed by the eukaryotic cell providesmolecular biologists with a useful tool for separation or selectiveisolation of poly A⁺ mRNAs from total cellular RNA. Separation of poly aA⁺ mRNAs from rRNA and tRNA can be achieved by hybridizing thepolyadenylated tails of mRNA molecules to oligo dT primers which arecoupled to a solid phase matrix. RNA species lacking poly A (rRNA andtRNA) fail to bind to oligo dT and are removed. Since high saltconditions are necessary to allow hybridization, the poly A⁺ mRNA cansubsequently be released by lowering the ionic strength anddestabilizing the dT:A hybrids.

Upon the poly A⁺ mRNA isolation, a cDNA library was constructed usingRT-PCR (reverse transcription—polymerase chain reaction) following theOmniscript Protocol for Reverse Transcription (Omniscript ReverseTranscriptase Handbook, 1999, the content of which we incorporate hereinby reference). Reverse transcriptase is a multifunctional enzyme withseveral distinct enzymatic activities, two of which, an RNA-dependantDNA polymerase and a hybrid-dependent exoribonuclease (RNase H), areutilized for reverse transcription in vitro to produce single-strandedcDNA with RNA as a starting template. The RNA-dependent DNA-polymeraseactivity (reverse transcription) transcribes cDNA from an RNA templatewhich allows synthesis of cDNA for subsequent PCR. An exoribonucleaseactivity (RNase H) of Omnicript Reverse Transcriptase specificallydegrades only the RNA in RNA:DNA hybrids. This Omniscript RNAse Hactivity affects RNA that is hybridized to cDNA and also improves thesensitivity of subsequent PCR.

The reverse-transcription (RT) reaction conditions were as follows:

10X Buffer RT 2.0 μL dNTP mix (5 mM each dNTP) 2.0 μL Oligo-dT primer(SEQ ID NO: 3) 10 μM 2.0 μL RNase inhibitor (10 units/μL) 1.0 μLOmniscript Reverse Transcriptase (4 units/μL) 1.0 μL RNase-free water9.0 μL Template poly A + RNA (˜25 ng/μL) 3.0 μL Total  20 μL

4. 60-Nucleotide Primer Used to Screen cDNA Library for Cocoon SilkProtein Transcript

Two primers may be preferably used to promote the building of a newstrand of DNA encoding the cocoon silk protein after DNA strands wereseparated by heating during the PCR process.

Primer #1, the cocoon silk protein primer, was a degenerate primer ofthe following structure:

5′ end

GGN GTN GCN CCN AAN AAN TAN CGN AAN GGN CAN TAN GTN SEQ ID NO: 2 GGN GGNTAN GGN AAN AAN TAN

Primer #2 was a poly-T primer of the following structure:

5′-TTTTGTACAAGCTT₃₀N₂-3′, SEQ ID NO: 3 where N can be any of A, T, G orC.

where N can be any of A, T, G or C.

The conditions of the polymerase chain reaction were as follows:

1. The PCR Mixture, Using the Qiagen kit, Catalogue No. 201203,Consisted of

Q-solution 10X 4 μL 10X PCR Buffer (with 15 Mm MgCl₂) 2 μL dNTPssolution containing 10 mM of each dNTP 2 μL MgCl₂ 25 mM 1 μL 10 μMOligo-dT primer (SEQ ID NO: 3) 1 μL 85 pmoles/μL cocoon silk proteinprimer (SEQ ID NO: 2) 0.4 μL Taq polymerase (5 units/μL) 0.2 μL Template(finished RT product, ˜25 ng/μL) 4 μL dH₂O 5.4 μL Total 20 μL

For PCR following RT, Omniscript recommends no more than ⅕ of the totalreaction volume should be derived from the finished RT product. Themaximum recommended was used, i.e. 4 μL of 20 μL.

2. The Thermocycler Program was as Follows

1) 95° C. 15 min 2) 94° C.  2 min 30 sec 3) 55° C.  3 min 4) 72° C.  2min 30 sec 5) 72° C.  5 min final extension

Steps 2-4 were run for 45 cycles. The sample was then run on an ethidiumbromide gel and a single band <750 bp was visualized.

5. Ligation of RT-PCR Product Using pGEM-T™ Vector System from Promega

The RT-PCR product of step 4 was then ligated preferably using pGEM-T™Easy Vector System from Promega (Cat. No. A3600). The resultant DNA fromthe RT-PCR reaction was purified using a GFX™ PCR DNA and Gel BandPurification kit (Amersham Pharmacia Biotech, Cat. No. 27-9602-01)according to manufacturer's instructions and eluted in 40 μL dH₂O. Theabove purification removes salts, enzyme, unincorporated nucleotides andpromoters from PCR products. The resulting concentration of RT-PCR DNAwas approximately 20 ng/μL. This purified RT-PCR DNA, approximately 0.7kb in length, was then used as an insert for ligation into a pGEM-T™Vector plasmid following the steps in “The Experienced User's Protocolfor Promega pGEM-T™ Vector Systems”, the content of which isincorporated herein by reference. The ligation mixture used was asfollows:

2X Rapid Ligation Buffer, T4 DNA ligase 5 μL pGEM-T vector (50 ng) 1 μLpurified RT-PCR DNA (20 ng/μL) 3 μL T4 DNA ligase (3 Weiss Unit/μL) 1 μLTotal 10 μL 

6. Transformation of E. coli XL1 Blue Cells

E. coli XL1 Blue cells were transformed with the ligation mixture ofstep 5 as follows. E. coli XL1 Blue cells (Stratagene) were madecompetent, i.e. those cells were treated to enhance their ability totake up DNA. Protocol to make cells competent was modified fromSambrook, J., Fritsch, E. F., and Maniatis, T., 1989, Molecular Cloning:A Laboratory Manual, 2^(nd) Edition, Cold Spring Harbor LaboratoryPress, the content of which we incorporate herein by reference. Theactual procedure for making E. coli XL1 Blue cells (Stratagene)competent was as follows.

E. coli strain XL1-Blue cells were grown for 18 hours in 5 ml of LBbroth at 37° C. and 250 rpm shaking (LB is Luria-Burtani Medium (pH 7.0)containing 2 g bacto-tryptone, 1 g bacto-yeast extract, 2 g NaCl in 200mL dH₂O). Then, 200 μl of the above mixture with E. coli cells wastransferred into 50 ml of new LB broth and grown for 3 hours at 37° C.and 250 rpm shaking. After that, the mixture was centrifuged at 7.5K rpmfor 3 minutes and supernatant was discarded. The cells were thenre-suspended in 5 ml of Buffer A. The composition of the Buffer A was asfollows: 100 mM NaCl, 5 mM MgCl₂, 5 mM Tris-HCl, pH 7.5. Re-suspended E.coli cells were incubated on ice for 10 minutes and centrifuged at 7.5Krpm for 3 minutes. After that, a supernatant was discarded and a residuere-suspended in 5 ml of Buffer B. The composition of the Buffer B was asfollows: 100 mM CaCl₂, 5 mM MgCl₂, 5 mM Tris-HCl, pH 7.5. The resultingmixture with E. coli cells was incubated on ice for 30 minutes and thecells became competent. 10 μL of the ligation mixture (step 5) was addedto 190 μL of the competent cells. The ligation mixture with thecompetent cells was incubated on ice for 1 hour, then subjected to aheat shock at 42° C. for 90 seconds, and then again incubated on ice for5 minutes. After that, 1 mL of LB broth was added and E. coli cells weregrown at 37° C. and 250 rpm shaking for one hour.

The resulting transformed cells were plated into LB/amp/IPTG/Xgalplates. LB/amp/IPTG is Luria-Burtani Medium containing 1.5% agar, 75μL/mL ampicillin, with each agar plate subsequently overlaid with 20 μLof a 100 mM solution of isopropyl-thio-beta-D-galactopyranoside in waterand 50 μL of a 2% solution of5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside in dimethylsulfoxide. This agar medium is referred to as LB/amp/IPTG/Xgal. Afterthat, E. coli colonies were screened to determine which coloniescontained plasmids with the desired DNA insert. The screening is basedon E. coli color change. E. coli that have been transformed with theplasmid that had the insert from a RT-PCR of step 3 and subsequent PCRof step 4 would be white. Those E. coli colonies that have beentransformed with a plasmid that did not contain the desired insert wouldbe blue. Several of the white colonies were tested to make sure thatthey did, in fact, contain the DNA insert in question.

7. Plasmid Preparation for Nucleotide Sequencing

To obtain the nucleotide sequence of the cocoon silk protein,plasmids-were first prepared for sequencing by selecting white coloniesfrom the cells of step 6, growing them overnight, and then putting themthrough the Biorad plasmid mini-prep kit (Cat. No. 732-6100). Eag 1 (NewEngland BioLabs, Cat. No. 505S) was preferably used as the restrictionenzyme to digest the plasmid to screen for an insert approximately600-700 bp in length.

8. Nucleotide Sequence

T7 and SP6 primers were preferably used to sequence the insert of step7. These primers were provided by the sequencing facility. Sequences forthe above primers are as follows:

Primer #3, T7 primer: 5′ TAA TACGA CTCAC TATAG GGCG A 3′ SEQ ID NO: 4Primer #4, SP6 primer: 5′ AT TTAGG TGACA CTATA GAATA C 3′ SEQ ID NO: 5The following nucleotide sequence of the insert of step 7 was derived:5′ end SEQ ID NO: 6                                      AG CTC TCC CATATG GTC GAC CTG CAG GCG GCC GCA CTA GTG ATTGGA GTT GCT CCA AAG AAG TAC CGC AAG GGA CAC TATGTC GGG GGT TAC GGG AAG AAG TAT CGT ATT TTT GACAGC AAT TGT GCT ATG AAC AAC GCC AAC TGT CAG AATCCA AAC GAA TCC GCC TTC GCC GAA GTT GAT TTC ACGCTG TGC AAT GAT ATC AAA TGT CCT AGG AAA TGC GATAAA AAA CTA GAC CCG GTT TGT GCT TTT GAT GGG AAAACG TAC AGA CAA TTT AAC AAC AAA TGT CTG CTG CAAGAA TTC AAT GAT TGC GAT CAA AAT GTG TTT CAA TATTTC AAC GCT GTG ACT AAC AAA AAA ATG TGC GTG GTTGAG AAG CCA AAA TGC CCG ACC ATT TGT CCA GCA ATTTAT GCT CCC GTT TGT GGT CGA AAT GCC AAA GGG GATTAC AAA AGT TTT GCG AGT GAA TGC AAC CAA TCC GCATTC AAC TGC TTG ATT TCT AAG AAT CAA TAT ACG GGCAAG TAT GAT TTG AGT TTT TGC GAC ATC GAG TTC CCT TAA GCA TGA CGT TGT AACGTT TTT TCT CTG GAT GTG CAA AAC ATA AAT TAC AAG CAC TGG ATT GAA TGG TGTTTT ATT AAA TTT CCT TGT GAC CTT TTT TCC ATT ATT CTT TCC GGC CTT TAA CAAGTA ATC AAT ATT GAT ATC GGT CGT TTT TGT AAA GAT TTT TTT TCA GTA AAA ATATCC ATC TCA TTT TCA CAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAG CTT GTACAA AAA ATC CCG CGG CCA TGG CGG CCG GGA GCA TGC GAC GTC GGG CCC A

The underlined section of the above sequence corresponds to the deducedreading frame of the black fly cocoon silk protein gene. In general, thededuced reading frame is the codon sequence that is determined byreading nucleotides in groups of three, starting from a specific startcodon. In this case, the initial amino acid sequence was determined fromN-terminal portion of the protein and this sequence then corresponded tothe nucleotide sequence when read in triplets (codons).

9. Complete Amino Acid Sequence for Cocoon Silk Protein (Deduced fromNucleotide Sequence)

The DNA sequence of step 8 (SEQ ID NO: 6) was assessed for stop codonsand the encoded amino acid sequence was deduced using all of theunderlined nucleotides as shown in SEQ ID No: 6. The amino acid sequencewas deduced to be as follows:

GVAPKKYRKGHYVGGYGKKYRIFDSNCAMNNANCQNPNESAFAEVDFTLCNDIKCPR SEQ ID NO: 7KCDKKLDPVCAFDGKTYRQFNNKCLLQEFNDCDQNVFQYFNAVTNKKMCVVEKPKCPTICPAIYAPVCGRNAKGDYKSFASECNQSAFNCLNSKNQYTGKYDLSFCDIEFP

Due to the redundancy of the genetic code, i.e. more than one nucleotidetriplet (codon) can code for a single amino acid, more than onenucleotide sequence can potentially code for cocoon silk protein.Therefore, various other homologues can code for cocoon silk protein.Homology refers to sequence similarity between two peptides or betweentwo nucleic acid molecules. Homology is determined by comparing aposition in each sequence which may be aligned for purposes ofcomparison. When a position in the compared sequence is occupied by thesame base or amino acid, then the molecules are homologous at thatposition.

Although the invention has been described with reference to certainspecific embodiments, various modifications thereof will be apparent tothose skilled in the art without departing from the spirit and scope ofthe invention as outlined in the claims appended hereto.

7 1 20 PRT Simulium vittatum 1 Gly Val Ala Pro Lys Lys Tyr Arg Lys GlyHis Tyr Val Gly Gly Tyr 1 5 10 15 Gly Lys Lys Tyr 20 2 60 DNA Simuliumvittatum misc_feature (1)..(60) n can be any of A, T, G or C 2ggngtngcnc cnaanaanta ncgnaanggn cantangtng gnggntangg naanaantan 60 345 DNA Simulium vittatum misc_feature (44)..(45) n can be any of A, T, Gor C 3 ttttgtacaa gctttttttt tttttttttt tttttttttt tttnn 45 4 23 DNA T7primer 4 taatacgact cactataggg cga 23 5 23 DNA SP6 primer 5 atttaggtgacactatagaa tac 23 6 831 DNA Simulium vittatum 6 agctctccca tatggtcgacctgcaggcgg ccgcactagt gattggagtt gctccaaaga 60 agtaccgcaa gggacactatgtcgggggtt acgggaagaa gtatcgtatt tttgacagca 120 attgtgctat gaacaacgccaactgtcaga atccaaacga atccgccttc gccgaagttg 180 atttcacgct gtgcaatgatatcaaatgtc ctaggaaatg cgataaaaaa ctagacccgg 240 tttgtgcttt tgatgggaaaacgtacagac aatttaacaa caaatgtctg ctgcaagaat 300 tcaatgattg cgatcaaaatgtgtttcaat atttcaacgc tgtgactaac aaaaaaatgt 360 gcgtggttga gaagccaaaatgcccgacca tttgtccagc aatttatgct cccgtttgtg 420 gtcgaaatgc caaaggggattacaaaagtt ttgcgagtga atgcaaccaa tccgcattca 480 actgcttgat ttctaagaatcaatatacgg gcaagtatga tttgagtttt tgcgacatcg 540 agttccctta agcatgacgttgtaacgttt tttctctgga tgtgcaaaac ataaattaca 600 agcactggat tgaatggtgttttattaaat ttccttgtga ccttttttcc attattcttt 660 ccggccttta acaagtaatcaatattgata tcggtcgttt ttgtaaagat tttttttcag 720 taaaaatatc catctcattttcacaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaagcttg 780 tacaaaaaat cccgcggccatggcggccgg gagcatgcga cgtcgggccc a 831 7 168 PRT Simulium vittatum 7 GlyVal Ala Pro Lys Lys Tyr Arg Lys Gly His Tyr Val Gly Gly Tyr 1 5 10 15Gly Lys Lys Tyr Arg Ile Phe Asp Ser Asn Cys Ala Met Asn Asn Ala 20 25 30Asn Cys Gln Asn Pro Asn Glu Ser Ala Phe Ala Glu Val Asp Phe Thr 35 40 45Leu Cys Asn Asp Ile Lys Cys Pro Arg Lys Cys Asp Lys Lys Leu Asp 50 55 60Pro Val Cys Ala Phe Asp Gly Lys Thr Tyr Arg Gln Phe Asn Asn Lys 65 70 7580 Cys Leu Leu Gln Glu Phe Asn Asp Cys Asp Gln Asn Val Phe Gln Tyr 85 9095 Phe Asn Ala Val Thr Asn Lys Lys Met Cys Val Val Glu Lys Pro Lys 100105 110 Cys Pro Thr Ile Cys Pro Ala Ile Tyr Ala Pro Val Cys Gly Arg Asn115 120 125 Ala Lys Gly Asp Tyr Lys Ser Phe Ala Ser Glu Cys Asn Gln SerAla 130 135 140 Phe Asn Cys Leu Asn Ser Lys Asn Gln Tyr Thr Gly Lys TyrAsp Leu 145 150 155 160 Ser Phe Cys Asp Ile Glu Phe Pro 165

The emodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
 1. An isolated polypeptide molecule having an amino acid sequence of SEQ ID NO:
 1. 2. An isolated polypeptide molecule having an amino acid sequence of SEQ ID NO:7.
 3. A fiber formed from the polypeptide of claim
 2. 4. A fiber formed from the polypeptide of claim
 1. 